This invention relates to improved methods for the identification of transformed plants. More specifically, it relates to a method which eliminates the need for use of ancillary nucleotide sequences, such as selectable markers, to identify successful transformation by providing a method for direct detection of the expression of nucleotide sequences of interest in regenerated plant tissue.
The publications, patents and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date.
Early transformation procedures resulted in very low frequency of transformation, where only about one out of every one million plant cells were transformed successfully. This very low frequency of transformation presented a problem in identifying the single cell that had been transformed among all of the cells that were not transformed. This identification problem has been addressed generally by physically linking ancillary nucleotide sequences, such as a bacterial gene that confers antibiotic resistance, to the gene of interest. This construct is then used to transform a culture of plant cells or protoplasts, and the linked gene for antibiotic resistance can be used as a selectable marker to identify successful transformation with the gene of interest. Cells that have been transformed with the gene of interest can be easily identified because the only plant cells able to survive are the plant cells containing the resistance gene (and linked gene of interest).
The presence of ancillary marker sequences, such as antibiotic resistance genes, have provided a practical solution to the task of identifying successful transformation events. However, these ancillary sequences do not positively contribute to the final cultivar and, in fact, they lessen its commercial desirability. The environmental and biological effects of the presence of this unwanted genetic material in the cultivar is unclear, and commercialization of transgenic plants has met resistance and skepticism in large part because of the uncertainty associated with the marker material sequences. The presence of these undesirable sequences may also complicate the regulatory procedures necessary to bring the cultivar to the marketplace. Thus, it would be desirable to devise an alternative method of identifying successful transformation events which is not based on the use of bacterial resistance genes or other ancillary sequences which remain in the cultivar.
Known methods for eliminating marker, or other ancillary, sequences from the transformed cultivar include the use of transposons. In this method, plants are first transformed by use of transposons, which include marker genes. The gene of interest is either included within a transposon or, alternatively, in a DNA construct which is not part of the transposon. Subsequent crosses are required in order to eliminate the marker sequences by selecting progeny in which they do not appear (U.S. Pat. No. 5,225,341, Yoder et al.; issued Jul. 6, 1993). Additional disadvantages of the use of markers include the need to segregate away the marker DNA and the DNA footprint that the transposon leaves in the plant genomic DNA upon excising. Although the marker sequences are not present in the mature plant, this method is also based on the use of marker sequences and requires culturing on selective medium as a method for identifying transformed plant cells prior to regeneration of the plant.
A reliable method for selection of transformed plants which does not require the use of transposons, insertion of ancillary marker sequences or culturing on selective media, would simplify the transformation process, increase public acceptance and avoid related regulatory problems.